SARS-CoV-2 Omicron potently neutralized by a novel antibody with unique Spike binding properties

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   ScreenIT

   Mar 21, 2022

   SciScore for 10.1101/2022.03.18.484873: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IRB: Study design and use of subject samples were approved by the Institutional Review Board of the Lausanne University Hospital and the ‘Commission d’éthique du Canton de Vaud’ (CER-VD).
   Sex as a biological variable	Four female cynomolgus macaques aged 3–6 years were randomly assigned between the control and treated groups to evaluate the efficacy of P2G3 LS in the prophylaxis challenge study.
   Randomization	Four female cynomolgus macaques aged 3–6 years were randomly assigned between the control and treated groups to evaluate the efficacy of P2G3 LS in the prophylaxis challenge study.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies

   SciScore for 10.1101/2022.03.18.484873: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IRB: Study design and use of subject samples were approved by the Institutional Review Board of the Lausanne University Hospital and the ‘Commission d’éthique du Canton de Vaud’ (CER-VD).
   Sex as a biological variable	Four female cynomolgus macaques aged 3–6 years were randomly assigned between the control and treated groups to evaluate the efficacy of P2G3 LS in the prophylaxis challenge study.
   Randomization	Four female cynomolgus macaques aged 3–6 years were randomly assigned between the control and treated groups to evaluate the efficacy of P2G3 LS in the prophylaxis challenge study.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Spike and RBD tetramers were prepared fresh before use and formed by combining biotinylated proteins with PE-conjugated Streptavidin (BD Biosciences) at a molar ratio of 4:1. Binding and ACE2 blocking studies with SARS-CoV-2 Spike: Luminex beads used for the serological and purified antibody binding assays were prepared by covalent coupling of SARS-CoV-2 proteins with MagPlex beads using the manufacturer’s protocol with a Bio-Plex Amine Coupling Kit (Bio-Rad, France).	ACE2 suggested: None
   Biotinylated P5C3, P2G3, REGN10933, REGN10987, AZD8895, AZD1061, ADG-2 or S309 antibodies (prepared as described above) were added to each well at 1 μg/ml followed by a further 20-minute incubation.	AZD8895 suggested: None S309 suggested: None
   Freshly isolated cells were stained with the cocktail of fluorescent conjugated antibodies containing anti-CD19 APC-Cy7, anti-CD3-BV510, anti-IgM-FITC, anti-IgD PE-CF594, anti-CD27-APC, anti-CD38-V450 (BD Biosciences) along with the pre-complexed Beta variant Spike tetramer (2 μg in 100μl) coupled to PE-streptavidin (BD Biosciences).	anti-CD19 APC-Cy7, anti-CD3-BV510, anti-IgM-FITC, anti-IgD PE-CF594 suggested: None anti-IgD suggested: None anti-CD27-APC suggested: (Sigma-Aldrich Cat# SAB4700132, RRID:AB_10896618) anti-CD38-V450 suggested: None
   The treated group (n = 2 [MF1 and MF2]) received one dose at 10 mg/kg of P2G3 LS human IgG1 monoclonal antibody delivered by intravenous injection three day prior to challenge, while control animals (n = 2 in parallel [MF3 and MF4] and n=2 historical [MF5 and MF6]) received no treatment.	human IgG1 suggested: None MF4 suggested: None
   In the ADCC assay, CEM-NKR-Spike-Luc cells were incubations with anti-Spike antibody at 0.3 μg/ml, isotype control antibodies at 0.3 μg/ml or an anti-HLA class I (MHC) positive control antibody (Invivogen) at 0.005 μg/ml.	anti-Spike suggested: None I (MHC) positive control antibody (Invivogen) at 0.005 suggested: None
   To monitor cell killing, co-cultured cells were washed and stained with fluorescent conjugated antibodies, CD56-AF488 (BD Biosciences,), CD16-FITC (BD Biosciences), Aqua Live/Dead cell stain (Invitrogen), CD4-PECF594 (BD Biosciences) and Annexin V-APC (BD Biosciences), and then analysed using a FACS LSR II cytometer instrument.	Annexin V-APC suggested: None
   Positive control anti-MHC (anti-HLA class I) antibody gave strong antibody dependent cell killing of 60-80% and anti-Spike antibodies gave intermediate responses.	anti-MHC suggested: None anti-HLA class I suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Viral stocks were prepared in EPISERF on VeroE6 or Calu-3 (for Omicron), aliquoted, frozen and titrated on VeroE6 cells.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Rev and pUltra-Chili-Luc vectors (Addgene) into 293T cells in DMEM medium + 10% FCS using Fugene 6 (Promega) for pseudoviruses production.	293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
   All animals were then exposed to a total dose of 105 TCID50 of Omicron B.1.1.529 SARS-CoV-2 virus produced in Calu-3 cells (NIH/BEI reference: NR-56462) via the combination of intranasal and intratracheal routes (day 0) with sample collection and testing performed as previously described 36.	Calu-3 suggested: None
   U937 cells incubated with Spike beads in the absence of antibody generally showed <5% phagocytic activity while increased ADCP activity was observed with increasing concentration of anti-Spike antibody with a maximum of 100% of cells exhibiting fluorescence associated with Spike bead phagocytosis.	U937 suggested: None
   Recombinant DNA
   Sentences	Resources
   The Omicron Spike ectodomain was amplified by PCR with primers (listed in Table 3) designed on consensus sequence from available Omicron sequences, and introduced by In-Fusion cloning into the nCoV-2P-F3CH2S plasmid, replacing the original wild-type Spike29.	nCoV-2P-F3CH2S suggested: None
   Pseudoviruses were alternatively produced with the original 2019-nCoV (Cat #100976), Alpha / B.1.1.7 (Cat #101023) and Beta/B.1.351 (Cat #101024) pCAGGS-SARS2-Spike vectors obtained from NIBSC.	pCAGGS-SARS2-Spike suggested: None
   These vectors were co-transfected with pMDL p.RRE, pRSV.	pMDL p.RRE suggested: None pRSV suggested: RRID:Addgene_106453)
   Rev and pUltra-Chili-Luc vectors (Addgene) into 293T cells in DMEM medium + 10% FCS using Fugene 6 (Promega) for pseudoviruses production.	pUltra-Chili-Luc suggested: RRID:Addgene_48688)
   Software and Algorithms
   Sentences	Resources
   To monitor cell killing, co-cultured cells were washed and stained with fluorescent conjugated antibodies, CD56-AF488 (BD Biosciences,), CD16-FITC (BD Biosciences), Aqua Live/Dead cell stain (Invitrogen), CD4-PECF594 (BD Biosciences) and Annexin V-APC (BD Biosciences), and then analysed using a FACS LSR II cytometer instrument.	BD Biosciences suggested: (BD Biosciences, RRID:SCR_013311)
   The soft mask volume were generated manually in UCSF Chimera and cryoSPARC 40.	cryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
   Cryo-electron microscopy model building: A model of a Spike trimer (PDB ID 7QO7) or AlphaFold2 (ColabFold implementation) models of the P5C3 and P2G3 Fabs were fit into the cryo-EM maps with UCSF Chimera These docked models were extended and rebuilt manually with refinement, using Coot and Phenix41,42.	Coot suggested: (Coot, RRID:SCR_014222)
   Buried surface area measurements and centroid measurements were calculated within ChimeraX.	ChimeraX suggested: (UCSF ChimeraX, RRID:SCR_015872)
   Analyses were performed in GraphPad Prism (GraphPad Software, Inc.) and Microsoft Excel.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798) Microsoft Excel suggested: (Microsoft Excel, RRID:SCR_016137)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

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   Results from scite Reference Check: We found no unreliable references.

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   Version published to 10.1101/2022.03.18.484873v1 on bioRxiv

   Mar 18, 2022